The long-term objective of this work is to understand the biochemistry of nuclear DNA replication. This extremely complicated process requires the coordinated activity of 3 DNA polymerises and multiple accessory proteins, and is a primary 2target for a variety of cancer chemotherapeutics. Thus, a greater understanding of the mechanism of DNA replication and how it can be inhibited may help lead to the development of novel chemotherapeutic strategies for the effective treatment of cancer. The enzyme complex of primary interest is DNA polymerase alpha-primase. On single-stranded DNA, primase synthesizes RNA primers that are further elongated by pol alpha via dNTP polymerization, a reaction sequence that is essential for initiation of all new strands of DNA. Primase consists of two subunits, p49 and p58, while pol alpha consists of a single subunit, p180. While the basic reactions catalyzed by both pol alpha and primase have been characterized, little is know about the individual subunits function together to catalyze the complicated set of reactions required to synthesize a new strand of DNA. To better understand this process, 3 specific aims will be undertaken. In the first two aims, the functions of the p58 and p49 primase subunits during primer synthesis and transfer of primers to pol alpha will be analyzed. Site-directed mutagenesis will be used to delete regions of each protein as well as change individual amino acids that are likely important for catalysis. The effects on primer synthesis and transfer of primers to pol alpha will be quantified using both steady state and pre-steady state kinetic methods. Changes in the binding of p49 and p58 to each other as well as to the p180 pol alpha subunit will also be measured. In the third aim, the interaction of pol alpha-primase with single-stranded DNA binding protein (RPA) will be examined. Interactions between RPA and the subunits of pol alpha-primase will be quantified, and both steady-state and pre-steady state kinetic methods will be used to understand how RPA regulates both pol alpha and primase activity. Together, the proposed studies will provide a detailed understanding of how pol alpha-primase initiates the synthesis of new strands of DNA. Additionally, the RPA studies will show how pol alpha-primase interacts with a protein that may be a key regulator of replication, and will provide a basis for understanding how pol alpha-primase interacts with the many other proteins present at replication forks.